Project Summary Stem cells replenish and rejuvenate tissue as an essential part of normal function. With age, this ability declines, resulting in effects such as cognitive impairment, reduced immune response, deterioration of skeletal muscle, and difficulty in wound healing. The mechanisms behind this dysfunction remain poorly understood. Similar to budding yeast, we have found a type of replicative aging in adult mouse neural stem cells (NSCs). In young NSCs, we identified an asymmetric segregation of ubiquitinated proteins and vimentin between daughter cells during mitosis, resulting in decreased proliferation of the receiving daughter. Concurrently, we found that young NSCs possess a diffusion barrier in the endoplasmic reticulum (ER) membrane, limiting the movement of proteins between daughter cells. Interestingly, NSCs from old mice have a weakened diffusion barrier, and more symmetric cargo segregation. In divisions resulting in asymmetric fate between daughters, the non-stem progeny inherits the cargoes. These studies imply that young NSCs may use asymmetric cargo segregation to rejuvenate themselves, and that loss of this ability with age greatly contributes to the stem cell aging phenotype in the body. Thus, we hypothesize that replicative aging at the stem cell level may lead to organismal aging. To address this hypothesis, we must first understand what mechanisms underlie asymmetric cargo segregation, determine what cargoes are segregated and their role, determine how these processes function in the adult brain, and test if this system can be targeted to improve stem cell aging. Previously, we found that disruption of the nuclear envelope (NE) weakens the diffusion barrier and leads to symmetric cargo segregation. Thus, we will target NE proteins to determine their role in cargo segregation. To identify novel candidates involved in asymmetry, we will use a CRISPR GeCKO library to screen mouse NSCs with fluorescently labeled cargoes, using automated microscopy to detect dividing cells and follow their division over time. We will identify mutations that affect asymmetry, and manipulate protein levels in the old brain to determine if this can rescue stem cell proliferation and neurogenesis. Additionally, we will identify other asymmetrically segregated cargoes, creating a map of their relative inheritance between daughter cells, and establishing the functional consequence of this inheritance. To reveal how the asymmetric segregation of cargoes works in the brain, we will create a transgenic mouse tagging endogenous vimentin with mNeon in NSCs and their progeny, to visualize these processes in the adult brain. We will determine if modulating levels of vimentin, ubiquitin, or newly identified cargoes in vivo, can rescue or maintain NSCs during aging. These experiments open up a new area of stem cell biology. Addressing these questions will establish a broad foundation for understanding asymmetric segregation and how aging at the cellular level relates to organismal aging. This work will lead to the identification of novel therapeutic targets for stem cell aging and stem cell-associated diseases.